Strong correlations exist between intra-uterine and/or fetal inflammation, prematurity and incidence of adverse neonatal outcomes, including cerebral palsy. If targeted interventions to improve outcome are to be developed we need to fully understand the molecular mechanisms which inadvertently cause fetal cell damage and tissue injury. Damage-associated molecular pattern molecules (DAMPs) are a pleiotropic group of intra-cellular proteins including high-mobility group box 1 protein (HMGB1) and members of the S100 calcium binding protein family (i.e., S100 A12, S100A8, S100B). When released into the extra-cellular compartment as a result of inflammation or oxidative stress, DAMPs become danger signals by activating endogenous receptors such as the receptor of advanced glycation end-products (RAGE). Engagement of RAGE leads to cellular dysfunction and injury driven by oxidative stress and sustained activity of nuclear factor-kappa B (NF:B). Using proteomics we discovered that S100A12 plays a key role in orchestrating the intra-amniotic inflammatory response to infection via RAGE activation. We have also shown that human fetuses with heightened inflammatory statuses have increased systemic levels of prototype DAMPs such as HMGB1 and S100B. In addition, in a mouse model of endotoxin induced fetal damage we demonstrated that maternal inflammation is associated with fetal oxidative stress and depletion of the intracellular antioxidant glutathione. In the same experimental model we further provided evidence that HMGB1 and RAGE and over-expressed in the liver and brain of the fetuses exposed to inflammation in utero. This body of knowledge, corroborated with the evidence that HMGB1 and S100 proteins are putative RAGE ligands, has led us to propose an active role for the DAMP- RAGE axis in inducing antenatal end-organ damage in the setting of intra-amniotic infection and prematurity. To test this hypothesis, three aims will be pursued: 1) Specific Aim 1 is geared to provide in vivo observational evidence that intra-uterine inflammation induces an imbalance in the redox homeostasis of the human fetus, causing release of DAMP proteins and antenatal fetal cellular damage via RAGE activation; 2) In Specific Aim 2 we will explore, in vitro, the mechanism and functional role of prototypical DAMPs and RAGE signaling in inducing cellular injury in a relevant bioassay. We will test the efficacy of anti-HMGB1 and anti-RAGE blocking antibodies or potential therapeutic agents such as N-acetylcysteine and ethyl pyruvate to reverse the damaging effects of the DAMP-RAGE axis activation; 3) In Specific Aim 3, by using unique genetically engineered RAGE deficient (RAGE-/-) and RAGE transgenic (RAGETg+) animals, we plan to provide insight into RAGE signaling as the common and obligatory pathway leading to fetal damage and explore the value of DAMP and RAGE antagonism as treatment strategies in vivo. By completing these aims we hope to provide novel insight into the processes leading to end-organ damage in premature infants and identify new classes of molecular targets that can modulate the inflammatory response of the fetus and improve outcomes. PUBLIC HEALTH RELEVANCE: Damage-associated molecular pattern molecules (DAMPs) and the receptor of advanced glycation end- products (RAGE) are endogenous mediators of cellular injury leading to irreversible tissue damage. We discovered that in pregnancies complicated by intra-amniotic infection, the heightened states of inflammation and oxidative stress have all the requisite attributes to trigger RAGE engagement in vital fetal organs that may explain the increased incidence of poor outcomes associated with prematurity. By using genetically engineered animal models, we aim to provide insight into the pathological consequences of fetal RAGE activation and the potential of RAGE targeted therapeutic interventions to lower the neonatal morbidity and mortality of premature infants.